A SPLICE-SITE MUTATION THAT INDUCES EXON SKIPPING AND REDUCTION IN LYSYL HYDROXYLASE MESSENGER-RNA LEVELS BUT DOES NOT CREATE A NONSENSE CODON IN EHLERS-DANLOS-SYNDROME TYPE-VI

The type VI variant of Ehlers-Danlos syndrome (EDS) is a heritable con nective tissue disorder caused by a deficiency in the activity of lysy l hydroxylase, an enzyme required for the post-translational processin g of collagens, We have characterized a novel type of mutation in a yo ung female patient with type VI EDS, in which cells possess only 12% o f the lysyl hydroxylase activity that is detected in unaffected cells, The syndrome was found to be caused by a homozygous insertion of two thymidines at the 5' splice site consensus sequence of intron 9 in the lysyl hydroxylase gene, The insertion interfered with normal splicing of the primary RNA transcript and resulted in an inframe deletion of the 132 nucleotides coded by exon 9 from the lysyl hydroxylase mRNA, I n addition, the mutation caused a marked reduction in the steady-state level of the truncated mRNA, which was less than 15% of the level fou nd in unaffected cells, The mutation also reduced the amount of the en zyme protein produced, which was estimated to be about 20% of that in control cells, However, the mutation did not affect the stability of t he abnormally spliced mRNA nor the normal localization of the enzyme p rotein in the endoplasmic reticulum, According to our results, the red uction in enzymatic activity observed in this patient is caused by low levels of both lysyl hydroxylase mRNA and enzyme protein, The primary cellular defect associated with this mutation, therefore, appears to be at the level of nuclear mRNA metabolism even though the mutation di d not create a premature translation termination codon.